In all domains of life, small RNAs (sRNAs) are vital regulators of normal cell function and responses to a variety of stresses. In bacteria, the mechanisms by which sRNAs regulate gene expression have been well- characterized, while related regulation and sRNA roles in stress are often poorly understood. The sRNA SgrS is a model for studying stress in Escherichia coli and other bacteria. SgrS and the transcriptional regulator SgrR are both essential for regulating the response to glucose-phosphate stress, which occurs when glucose- phosphate accrues in the cell and impedes growth. SgrR activates expression of SgrS, which helps rescue cells from stress largely by inhibiting translation of mRNAs that encode sugar transporters. While regulation by SgrS is well-described, little is known about regulation by SgrR or other associated regulatory networks. The long-term objectives of this proposal are to characterize the regulation and physiological functions of the glucose-phosphate stress response. In particular, Aim 1 will characterize novel genes regulated by SgrR and their role in glucose-phosphate stress, while Aim 2 will elucidate the regulatory relationship of SgrR-SgrS to the stringent response to nutrient starvation. For Aim 1, preliminary experiments utilizing high-throughput RNA sequencing (RNA-seq) analysis have identified dozens of novel gene targets potentially regulated by SgrR. We will verify these SgrR-mediated changes in expression using quantitative PCR (qPCR), and assess SgrR binding to target gene promoters using in vivo titration assays. To examine the functions of target genes in glucose-phosphate stress, we will delete and overexpress the genes and assess the effects on induction of the stress response and growth during stress. For Aim 2, previous findings strongly support the notion that glucose-phosphate stress is due to glycolytic or related nutrient depletion similar to stringent nutrient starvation. In addition, preliminary data support likely roles for stringent response regulators DksA and the alarmone ppGpp (synthesized by RelA and SpoT) in glucose-phosphate stress, as mutations in dksA or relA spoT result in growth defects during stress. We will further examine the role of dksA and ppGpp null mutations in GP stress response regulation as measured by expression of a PsgrS-lacZ transcriptional fusion. To determine whether the glucose-phosphate stress response affects stringent induction, we will examine the effects of mutating sgrR and sgrS on stringent response induction and growth under stringent conditions. Finally, to identify individual stringent response genes with functions in glucose-phosphate stress, we will use RNA-seq to monitor gene expression of dksA and ppGpp null mutants grown under glucose-phosphate stress. As with potential SgrR targets, we will then employ genetic analyses to assess stress-related phenotypes of stringent gene mutations. This work will aid in our understanding of how cells use sRNAs related regulators of their physiology in order to adapt to stress. Both sRNAs and sugar-phosphate stress play a role in virulence of certain bacteria, so this research also could ultimately contribute to finding new targets for treating disease.